Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

A process for preparing a lyophilised vaccine antigen, comprising steps
of (i) increasing the concentration of an antigen in a liquid composition
including that antigen using centrifugal filtration and/or
ultrafiltration, to provide a concentrated antigen, and (ii) lyophilising
the concentrated antigen, to provide the lyophilised vaccine antigen. The
lyophilised material can be reconstituted and used for vaccine
formulation. The process is particularly useful with influenza vaccine
antigens.

Claims:

1. A process for preparing a lyophillsed vaccine antigen, comprising
steps of (i) increasing the concentration of an antigen in a liquid
composition including that antigen using centrifugal filtration, to
provide a concentrated antigen, and (ii) lyophilising the concentrated
antigen, to provide the lyophilised vaccine antigen.

2. A process for preparing a reconstituted liquid vaccine antigen,
comprising steps of: (a) lyophilising an antigen by the process of claim
1; and (b) reconstituting the lyophilised vaccine antigen in an aqueous
liquid.

3. The process of claim 2, wherein the volume of the aqueous liquid used
in step (b) is lower than the volume of the liquid composition used at
the start of step (a).

4. The process of claim 2, wherein the volume of the aqueous liquid used
in step (b) is lower than the volume of the concentrated antigen made
during step (a).

5. The process of claim 2, wherein the reconstituted liquid vaccine
antigen is used to formulate a vaccine.

6. The process of claim 1, wherein the vaccine antigen is to protect
against disease caused by a bacterium, a virus, a fungus, and/or a
parasite.

7. The process of claim 5, wherein the vaccine is an influenza vaccine.

8. The process of claim 7, wherein the influenza vaccine is an
inactivated influenza vaccine.

13. The process of claim 12, wherein the microneedles are fabricated by
(a) mixing a biosoluble and biodegradable matrix material with the
reconstituted liquid vaccine antigen; and (b) adding the mixture from
step (a) to a mold containing cavities for forming microneedles.

15. The process of claim 14, wherein the microneedle is metal or plastic.

16. The process of claim 14, comprising applying the reconstituted liquid
vaccine antigen to the surface of one or more solid microneedles to
provide a coated microneedle device for injection of the vaccine.

17. The process of claim 12, wherein the microneedles are 100-2500 μm
long.

19. The process of claim 18, comprising mixing the reconstituted liquid
antigen with one or more orally-soluble polymers, then forming a film
using the mixture to provide a thin film suitable for buccal
administration of the vaccine.

20. The process of claim 18, comprising mixing the reconstituted liquid
antigen with one or more topically-soluble polymers, then forming a film
using the mixture to provide a thin film suitable for transcutaneous
administration of the vaccine.

21. The process of claim 18, wherein the film is 10-500 μm (e.g.
75-150 μm) thick.

22. The process of claim 18, wherein the antigen is encapsulated inside
microparticles within the film.

23. A process for preparing a packaged vaccine, comprising: (i) preparing
a solid vaccine by the process of claim 11; then (ii) packaging a solid
vaccine into an individual unit dose pouch.

24. A vaccine prepared by the process of claim 1.

25. A method of raising an immune response in a subject, comprising the
step of administering the vaccine of claim 24 to the subject.

26. A process for preparing a vaccine antigen, comprising steps of (i)
increasing the concentration of an antigen in a liquid composition
including that antigen, to provide a concentrated antigen, (ii)
lyophilising the concentrated antigen, to provide the lyophilised vaccine
antigen, and (iii) reconstituting the lyophilised vaccine antigen in an
aqueous buffer to provide a reconstituted antigen.

28. The process of claim 26, where reconstitution in step (iii) uses a
phosphate buffer.

Description:

[0001] This application claims the benefit of U.S. provisional application
61/396,720 (filed 1 Jun. 2010), the complete contents of which are hereby
incorporated herein by reference for all purposes.

TECHNICAL FIELD

[0002] This invention is in the field of processing antigen solutions for
use in vaccines.

BACKGROUND ART

[0003] During vaccine manufacture it is often the case that the
concentration of antigen in a manufacturing bulk exceeds the
concentration in a final patient formulation, and so the process involves
a step in which the bulk is diluted. In some situations, however, it is
necessary to increase the antigen concentration in an aqueous bulk, and
the invention concerns processes for concentrating antigens. Useful
processes should increase an antigen's concentration without destroying
its immunogenicity.

[0004] One situation where antigen concentration is required is for new
delivery techniques where only a small volume of material is delivered.
For instance, vaccines can be delivered by microneedles [2,3] or by thin
films or strips [1,14-17]. These techniques deliver much less volume than
the typical intramuscular injection of 0.5 ml but they may require the
same amount of antigen, which will often require a more concentrated bulk
antigen.

[0005] One existing concentration process which can increase the
concentration of an individual influenza virus hemagglutinin (HA) from
125-500 μg/ml to 14 mg/ml involves tangential flow filtration (TFF) of
a starting volume of aqueous material to a concentration of 10 mg/ml,
then lyophilisation, then reconstitution of the lyophilisate in a smaller
aqueous volume than the starting volume. This process can be performed on
three different monovalent HA bulks, and their reconstitution as a single
trivalent aqueous composition can provide a final HA concentration of 42
mg/ml.

[0006] It is an object of the invention to provide further and improved
processes for increasing the concentration of antigen in a material for
use in vaccine manufacture, and particularly for influenza vaccine
manufacture, such as influenza vaccines which are not delivered by
intramuscular injection.

DISCLOSURE OF THE INVENTION

[0007] In contrast to an existing process in which antigen is concentrated
using TFF, the antigen concentration procedure of the invention uses
centrifugal filtration and/or ultrafiltration. Like the existing process,
the concentrated material can then be lyophilised, and the lyophilised
material can be reconstituted for further use.

[0008] Thus the invention provides a process for preparing a lyophilised
vaccine antigen, comprising steps of (i) increasing the concentration of
an antigen in a liquid composition including that antigen using
centrifugal filtration and/or ultrafiltration, to provide a concentrated
antigen, and (ii) lyophilising the concentrated antigen, to provide the
lyophilised vaccine antigen.

[0009] The invention also provides a lyophilised vaccine antigen prepared
by this process.

[0010] The lyophilised vaccine antigen can be used to formulate a vaccine,
or can be reconstituted and then used to formulate a vaccine. This
reconstitution is ideally in a smaller volume than the liquid
composition's original volume, i.e. the volume at the start of step (i),
and smaller than the concentrated antigen's pre-lyophilisation volume,
i.e. the volume at the start of step (ii), as this again increases the
antigen concentration. The reconstituted material can be used to
formulate a vaccine.

[0011] The invention also provides a vaccine formulated by this process.

[0012] The process is particularly useful for preparing a lyophilised
influenza vaccine antigen, and this lyophilised influenza vaccine antigen
is useful for formulating influenza vaccines.

[0013] The Antigen

[0014] The invention is useful for concentrating antigens from various
sources. The antigen may be from a bacterium, a virus, a fungus, or a
parasite. Thus the vaccine may protect against disease caused by a
bacterium, a virus, a fungus, and/or a parasite.

[0015] Typical bacteria for use with the invention include, but are not
limited to:

[0024] Typical viruses for use with the invention include, but are not
limited to:

[0025] Orthomyxovirus, such as an influenza A. B or C
virus. Influenza A or B viruses may be interpandemic (annual/seasonal)
strains, or from strains with the potential to cause a pandemic outbreak
(i.e., influenza strains with new hemagglutinin compared to a
hemagglutinin in currently circulating strains, or influenza strains
which are pathogenic in avian subjects and have the potential to be
transmitted horizontally in the human population, or influenza strains
which are pathogenic to humans). Depending on the particular season and
on the nature of the strain, an influenza A virus may be derived from one
or more of the following hemagglutinin subtypes: H1, H2, H3, H4, H5, H6,
H7,H8, H9, H10, H11, H12, H13, H14, H15 or H16. More details are given
below.

[0029] Picornavirus, such as
Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and
Aphthoviruses. Enteroviruses include Poliovirus types I, 2 or 3,
Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6,
Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 and
Enterovirus 68 to 71. Preferably, the Enterovirus is poliovirus e.g. a
type 1 strain such as Mahoney or Brunenders, a type 2 strain such as
MEF-I, or a type 3 strain such as Saukett. An example of a Hepamaviruses
(also named Hepatoviruses) is Hepatitis A virus.

[0030] Togavirus, such
as a Rubivirus, an Alphavirus, or an Arterivirus. Rubiviruses, such as
Rubella virus, are preferred. Useful alphaviruses for inactivation
include aquatic alphaviruses, such as salmon pancreas disease virus and
sleeping disease virus.

[0044] The invention is ideal for preparing vaccines for viruses, and in
particular viruses where the vaccine antigen is a viral surface
glycoprotein. Thus the invention is ideal for concentrating influenza
virus hemagglutinin for preparing influenza vaccines, as described below
in more detail. Steps (1) and (ii), followed by reconstitution, can
provide an influenza vaccine antigen with a HA content of >5 mg/ml,
and even >10 mg/ml.

[0045] The Liquid Composition

[0046] A process of the invention increases the concentration of an
antigen in a liquid composition, thereby providing a concentrated antigen
for formulation purposes.

[0047] A preferred liquid composition is one which has never been
lyophilised before step (ii). A preferred liquid composition is
substantially free from lyoproteetants at the start of step (i). Thus a
composition may be substantially free from exogenous sugar alcohols (in
particular: sorbitol, mannitol, maltitol, erythritol, xylitol) and/or
from exogenous disaccharides (in particular: sucrose, trehalose, maltose,
lactulose, lactose, cellobiose). The combined concentration of (sorbitol,
mannitol, maltitol, erythritol, xylitol, sucrose, trehalose, maltose,
lactulose, lactose, cellobiose) in a liquid composition may thus be less
than 10 mg/ml (i.e. less than 1%) and is ideally less than 1 mg/ml e.g
less than 0.1 mg/ml.

[0049] The liquid composition may be monovalent (i.e. containing vaccine
antigen for protecting against only one pathogen) or multivalent (i.e.
containing vaccine antigen for protecting against more than one pathogen,
which includes where there is more than one different
non-cross-protective pathogen eg multiple meningococcal serogroups, or
multiple influenza A virus hemagglutinin types).

[0050] The invention can be used with liquid samples having a variety of
vaccine antigen concentrations. Typically the liquid sample will include
a vaccine antigen at a concentration of at least 1 μg/ml.

[0051] The Concentration Step

[0052] A process of the invention involves a step in which the
concentration olan antigen is increased using centrifugal filtration
and/or ultrafiltration.

[0053] Centrifugal filtration involves centrifugation of a liquid through
a filter. The filter retains the antigen to be concentrated but does not
retain solvent or smaller solutes. As the volume of the filtrate
increases, the concentration of the antigen in the retentate also
increases. This technique typically uses a fixed angle rotor. Various
suitable centrifugal filtration devices are commercially available e.g.
the products sold under trade marks Centricon®, Vivaspin® and
Spintek®. The cut-off of the filter will be selected such that the
antigen of interest remains in the retentate.

[0054] Ultrafiltration involves the use of hydrostatic pressure to force a
liquid against a semipermeable membrane. The filter retains the antigen
to be concentrated but does not retain solvent or smaller solutes.
Continued application of hydrostatic pressure causes the volume of the
filtrate to increase, and thus the concentration of the antigen in the
retentate also increases. Many ultrafiltration membranes are commercially
available. The molecular weight cut-off (MWCO) of an ultrafiltration
membrane determines which salutes can pass through the membrane (i.e.
into the filtrate) and which are retained (i.e. in the retentate). The
MWCO of the filter used with the invention will be selected such that
substantially all of the antigen of interest remains in the retentate.

[0057] After antigen concentration, a process of the invention lyophilises
the concentrated antigen to provide a lyophilised vaccine antigen.

[0058] Lyophilisation typically involves three stages within a chamber:
(a) freezing; (b) primary drying; and (c) secondary drying. Step (a)
freezes the mobile water of the conjugate. In step (b) the chamber
pressure is reduced (e.g. to ≦0.1 Torr) and heat is applied to the
product to cause the frozen water to sublime. In step (c) the temperature
is increased to desorb any bound water, such as water of crystallisation,
until the residual water content falls to the desired level.

[0059] An initial step in a typical lyophilisation, before freezing
occurs, is addition of a lyoprotectant. In some embodiments a
lyoprotectant may have been added prior to concentration in step (i), but
it is preferred to add it instead after concentration has occurred i.e.
at the end of step (i) or at the start of step (ii). This makes it easier
to control the amount of lyoprotectant which is present at the start of
lyophilisation freezing.

[0060] Thus a process of the invention may involve a step of adding one or
more lyoprotectants to the concentrated antigen. Suitable lyoprotectants
include, but are not limited to, sugar alcohols (such as sorbitol,
mannitol, maltitol, erythritol, xylitol) and disaccharides (such as
sucrose, trehalose, maltose, lactulose, lactose, cellobiose). Sucrose and
mannitol (or a mixture thereof) are preferred lyoprotectants for use with
the invention.

[0061] After lyophilisation, a lyophilised vaccine antigen can be
reconstituted. This reconstitution can use water (e.g. water for
injection, wfi) or buffer (e.g. a phosphate buffer, a Tris buffer, a
borate buffer, a succinate buffer, a histidine buffer, or a citrate
buffer). Buffers will typically be included in the 5-20 mM range. A
phosphate buffer is preferred.

[0062] Step (i) concentrated the a first liquid volume of vaccine antigen,
providing a composition with the same amount of antigen in a second
(reduced) liquid volume. Step (ii) dried this concentrated material. This
dried material can be reconstituted in a third liquid volume. If the
third volume is greater than the first volume, the overall process has
failed to concentrate the antigen. Similarly, if the third volume is
greater than the second volume, the reconstitution step has gone
backwards in terms of concentration. Thus the third volume is either
equal to or, preferably, less than the second volume. Thus the
lyophilisation/reconstitution steps can achieve a further antigen
concentration. Embodiments where the third volume is equal to (or greater
than) then second volume are still useful e.g. for buffer exchange, etc.,
but they are not preferred.

[0063] Formulation

[0064] Lyophilised vaccine antigen can be used to formulate a vaccine, but
will typically be reconstituted before doing so.

[0065] The invention can be used for preparing various vaccine
formulations. The increased antigen concentration means that the
invention is ideal for techniques which involve the delivery of small
volumes of material to a patient. For instance, the invention is useful
for preparing liquid vaccine formulations which have a unit dose volume
of 0.1 ml or less (e.g. for intradermal injection). The invention is also
useful for preparing solid (including solid non-lyophilised) vaccine
formulations, as these can require high antigen concentrations. As
described in more detail below, suitable solid formulations include, but
are not limited to, solid biodegradable microneedles, coated
microneedles, and thin oral films. Thus a formulation step in a process
of the invention may comprise: preparing a solid vaccine form from the
lyophilised vaccine antigen.

[0066] Formulated vaccines of the invention will retain lyoprotectant(s)
from the lyophilisation step. Thus a vaccine may comprise, for example,
one or more of sorbitol, mannitol, maltitol, erythritol, xylitol,
sucrose, trehalose, maltose, lactulose, lactose, and/or cellobiose.

[0067] Vaccines of the invention are ideally free from inulin.

[0068] Solid Biodegradable Microneedles

[0069] One useful solid formulation which can be prepared using the
invention is a solid biodegradable microneedle. These are typically not
administered alone but, rather, multiple needles are administered
simultaneously e.g. as a skin patch comprising a plurality of
microneedles.

[0070] The microneedles are solid, such that they retain their structural
integrity during storage and can penetrate a subject's skin when the
patch is applied. The mechanical characteristics which are required for
skin penetration depend on the organism in question, but they will
usually have sufficient strength to penetrate human skin. Materials for
forming suitable solid needles are readily available and these can be
tested to determine appropriate concentrations etc. for any particular
need.

[0071] The microneedles are biosoluble and biodegradable. Thus the solid
material dissolves in the skin after the patch is applied, in contrast to
the coated microneedles used in references 2 & 3 (see below). Having
dissolved, the material will then be metabolised to give harmless
end-products. The timescale for dissolving after applying the patch can
vary, but dissolving will typically commence immediately after applying
the patch (e.g. within 10 seconds) and may continue for e.g. up to 1
minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours,
10 hours, or 24 hours, until the microneedle has fully dissolved.
Materials with suitable in vivo dissolving kinetics are readily available
and these can be varied and tested to determine appropriate
concentrations etc. for any desired dissolution profile.

[0072] Suitable matrix materials for forming the microneedles will
typically be biosoluble and biodegradable polymers, and these may
comprise one or more carbohydrates. For example, the material may
comprise a cellulose, a dextrin, a dextran, a disaccharide, a chitosan, a
chitin, etc., or mixtures thereof. Other GRAS materials may also be used.
These materials can conveniently be combined with the vaccine antigen by
including them in the liquid used to reconstitute the lyophilised vaccine
antigen.

[0074] The microneedles can penetrate the skin. They should be long enough
to penetrate through the epidermis to deliver material into the dermis
(i.e. intradermal delivery), but are ideally not so long that they can
penetrate into or past the hypodermis. They will typically be 100-2500
μm long e.g. between 1250-1750 μm long, or about 1500 μm. At the
time of delivery the tip may penetrate the dermis, but the base of the
needle may remain in the epidermis.

[0075] The microneedles can have various shapes and geometries. They will
typically be tapered with a skin-facing point e.g. shaped as pyramids or
cones. A tapered microneedle with a widest diameter of <500 μm is
typical.

[0076] A single patch will typically include a plurality of microneedles
e.g. ≧10, ≧20, ≧30, ≧40, ≧50,
≧60, ≧70, ≧80, ≧90, ≧100, ≧200,
≧300, ≧400, ≧50, ≧750, ≧1000 or more
per patch. Where a patch includes a plurality of microneedles, it may
comprise a backing layer to which all of the microneedles are attached. A
unitary backing layer with ≧20 projecting microneedles is typical.
Where a patch includes a plurality of microneedles, these can be arranged
in a regular repeating pattern or array, or may be arranged irregularly.

[0077] A patch will typically have an area of 3 cm2 or less, for
example <2 cm2 or <1 cm2. A circular patch with a
diameter of between 0.5 cm and 1.5 cm is useful.

[0079] A patch of the invention has a skin-facing inner face and an
environment-facing outer face. The inner face may include an adhesive to
facilitate adherence to a subject's skin. When present, it is preferably
not present on the microneedles themselves i.e. the microneedles are
adhesive-free. Rather than have adhesive on the inner face, a patch may
have an additional backing which provides an outer adhesive margin for
adhering the patch to skin e.g. as seen in sticking plasters or nicotine
patches.

[0080] Patches as described above can be made by following the techniques
and guidance in references 4-8. For instance, a mold with 1.5 mm-long
microneedle cavities can be prepared. A matrix material of dextrin and
trehalose can be combined with an influenza vaccine and this aqueous
material can be centrifugally cast in the mold to form an array of solid
microneedles. A cellulose gel can then be cast over the matrix/vaccine
mixture (e.g. which mixture has formed a film) to form a backing layer on
the patch. When this backing layer has dried, it can be removed to give a
patch from which the solid microneedles project. Thus a formulation step
in a process of the invention may comprise: (a) mixing a biosoluble and
biodegradable matrix material with the vaccine antigen, usually by
reconstituting a lyophilised vaccine antigen; and (b) adding the mixture
from step (a) to a mold containing cavities for forming microneedles. It
may further comprise: (c) letting the mixture set in the mold, to form
solid microneedles; (d) optionally, applying material to the set
microneedles to provide a backing layer; and (e) removing the
microneedles (and optional backing layer) from the mold.

[0081] Patches may be packaged into individual pouches e.g. sealed under
nitrogen, then heat sealed. They should be stored carefully to avoid
damage to the microneedles.

[0082] Coated Microneedles

[0083] Another useful solid formulation which can be prepared using the
invention is a coated microneedle. These are typically not administered
alone but, rather, multiple needles are administered simultaneously e.g.
via a plurality of microneedles. One suitable product is marketed under
the trade name of Macroflux® (Zosano).

[0084] The microneedles are solid, such that they retain their structural
integrity during storage and can penetrate a subject's skin. The
mechanical characteristics which are required for skin penetration depend
on the organism in question, but they will usually have sufficient
strength to penetrate human skin. The microneedles are solid and remain
intact after insertion into a patient's skin (in contrast to the
biodegradable microneedles discussed above). Materials for forming
suitable solid needles are readily available and these can be tested and
selected for any particular need e.g. metals (such as stainless steel) or
polymers (such as polycarbonate, ideally medical grade). Metal needles
can be fabricated by using laser cutting and electro-polishing [9].
Polymer needles can be fabricated by microreplication and/or micromolding
(including injection molding). Suitable microneedles are disclosed in
references 2, 3, and 9-13.

[0085] An antigen of the invention can be coated onto the microneedles.
This coating can be achieved by a simple process such as dip-coating e.g.
involving a dipping step then a drying step (e.g. by evaporation), with
repetition as required. Other useful coating techniques are disclosed in
reference 11. Thus a formulation step in a process of the invention may
comprise: applying the lyophilised vaccine antigen, or a reconstituted
form thereof, to the surface of one or more solid microneedles to provide
a coated microneedle device for injection of the vaccine.

[0086] A coating solution for applying to the needles can include one or
more biosoluble and biodegradable matrix materials, and these may
comprise one or more carbohydrates. For example, the material may
comprise a cellulose, a dextrin, a dextran, a disaccharide, a chitosan, a
chitin, etc., or mixtures thereof. Other GRAS materials may also be used.
Suitable celluloses, dextrins and disaccharides are listed above. These
materials can conveniently be combined with the vaccine antigen by
including them in the liquid used to reconstitute the lyophilised vaccine
antigen.

[0087] Thus a formulation step in a process of the invention may comprise:
(a) mixing a biosoluble and biodegradable matrix material with the
vaccine antigen, usually by reconstituting a lyophilised vaccine antigen;
and (b) applying the mixture from step (a) to the surface of one or more
solid microneedles to provide a coated microneedle device for injection
of the vaccine. Coating may be enhanced by using one or more "deposition
enhancing components" as described in reference 11.

[0088] The applying steps discussed above may comprise an application
sub-step followed by a drying sub-step, and this pair of sub-steps can be
performed once or more than once e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more times.

[0089] Microneedles in the device can penetrate the skin when applied.
They should be long enough to penetrate through the epidermis to deliver
material into the dermis (i.e. intradermal delivery), but are ideally not
so long that they can penetrate into or past the hypodermis. They will
typically be 100-2500 μm long e.g. between 250-750 μm long, or
about 1500 μm. At the time of delivery the tip may penetrate the
dermis, but the base of the needle may remain in the epidermis. The
needles can be applied to a patient's skin for between 30 seconds and 30
minutes, and then be removed.

[0090] The microneedles can have various shapes and geometries. They will
typically be tapered with a skin-facing point e.g. shaped as pyramids or
cones. A tapered microneedle with a widest diameter of <500 μm is
typical.

[0091] A microneedle device will typically include a plurality of
microneedles e.g. ≧10, ≧20, ≧30, ≧40,
≧50, ≧60, ≧70, ≧80, ≧90, ≧100,
≧200, ≧300, ≧400, ≧50, ≧750,
≧1000, ≧1500, ≧2000 or more per device (for example,
300-1500 per device). Where a device includes a plurality of
microneedles, these will typically all be attached to a unitary backing
layer. Where a device includes a plurality of microneedles, these can be
arranged in a regular repeating pattern or array, or may be arranged
irregularly.

[0092] A microneedle device will typically have an area of 3 cm2 or
less, for example <2 cm2 or <1 cm2. A circular device
with a diameter of between 0.5 cm and 1.5 cm is useful.

[0093] The density of microneedles can vary, but may be ≧10
cm-2, ≧20 cm-2, ≧30 cm-2, ≧40
cm-2, ≧50 cm-2, ≧60 cm-2, ≧70
cm-2, ≧80 cm-2 or more. A device with 2 mm between each
microneedle, and a density of 14 microneedles/cm2, is useful.

[0094] A microneedle device has a skin-facing inner face and an
environment-facing outer face. The inner face may include an adhesive to
facilitate adherence to a subject's skin. When present, it is preferably
not present on the microneedles themselves i.e. the microneedles are
adhesive-free. Rather than have adhesive on the inner face, a device may
have an additional backing which provides an outer adhesive margin for
adhering the device to skin.

[0095] A microneedle device may be packaged into individual pouches e.g.
sealed under nitrogen, then heat sealed. They should be stored carefully
to avoid damage to the microneedles.

[0096] Thin Films

[0097] Another useful solid formulation which can be prepared using the
invention is a thin film, such as a thin oral film. These films wet and
dissolve quickly upon contact with saliva and buccal tissue, therefore
releasing the vaccine antigen in the mouth. The main component of these
thin films is typically one or more hydrophilic polymer(s), which can
have good mucoadhesive properties to provide strong adhesion to buccal
tissue until complete dissolution. Similar films can be used for non-oral
delivery e.g. for transcutaneous delivery as disclosed in reference 14.

[0098] Suitable thin films are typically 10-500 μm thick when initially
applied e.g. 75-150 μm thick. Their other dimensions can be suitable
to fit into a patient's mouth e.g. into an adult human mouth or into am
infant human mouth.

[0099] One suitable type of film is disclosed in reference 15. This film
comprises a mucoadhesive bilayer film with (i) Noveon and Eudragit S-100
as a mucoadhesive layer and (ii) a pharmaceutical wax as an impermeable
backing layer. Further details of these films are in reference 16.

[0101] The film in reference 14 comprises a cationic poly(-amino ester)
for transcutaneous delivery.

[0102] An oral film useful with the invention may include a flavouring
agent to make the vaccine more palatable during administration.

[0103] Thin films can be made a variety of processes, including but not
limited to: solvent casting; hot-melt extrusion; solid dispersion
extrusion; and rolling.

[0104] A formulation step in a process of the invention may thus comprise:
(a) mixing the vaccine antigen, usually by reconstituting a lyophilised
vaccine antigen, with one or more orally-soluble polymers; and (b)
forming a film using the mixture from step (a) to provide a thin film
suitable for buccal administration of the vaccine.

[0105] A formulation step in a process of the invention may comprise: (a)
mixing the vaccine antigen, usually by reconstituting a lyophilised
vaccine antigen, with one or more topically-soluble polymers, such as a
poly(β-amino ester); and (b) forming a film using the mixture from
step (a) to provide a thin film suitable for transcutaneous
administration of the vaccine.

[0106] These films may be packaged into individual unit dose pouches e.g.
sealed under nitrogen, then heat sealed. The pouches should be
water-tight to keep the films dry during storage.

[0107] Methods of Treatment, and Administration of the Vaccine

[0108] Formulated vaccines of the invention can be delivered to a subject
e.g. via their skin, via their buccal tissue, etc. Thus the invention
provides a method of raising an immune response in a subject, comprising
the step of administering a formulated vaccine of the invention to the
subject. This might involve e.g. applying a microneedle patch or device
to the subject's skin, such that the microneedles penetrate the subject's
dermis, or applying a thin film to the subject's buccal tissue or tongue.

[0109] The invention also provides a lyophilised antigen for use in a
method of vaccinating a subject. The invention also provides the use of
lyophilised antigen in the manufacture of a medicament for raising an
immune response in a subject.

[0110] The invention also provides a reconstituted lyophilised antigen for
use in a method of vaccinating a subject. The invention also provides the
use of reconstituted lyophilised antigen in the manufacture of a
medicament for raising an immune response in a subject.

[0112] The immune response raised by these methods and uses will generally
include an antibody response, preferably a protective antibody response.

[0113] Microneedle patches or devices may be applied to the skin by simple
manual application (e.g. as with a sticking plaster or with known skin
patches) or may be applied using a spring-driven injector.

[0114] Vaccines prepared according to the invention may be used to treat
both children and adults.

[0115] Treatment can be by a single dose schedule or a multiple dose
schedule. Multiple doses may be used in a primary immunisation schedule
and/or in a booster immunisation schedule. Multiple doses will typically
be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks,
about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12
weeks, about 16 weeks, etc.).

[0116] Influenza Vaccination

[0117] Processes of the invention are ideal for preparing influenza
vaccines. Various forms of influenza virus vaccine are currently
available (e.g. see chapters 17 & 18 of reference 18) and current
vaccines are based either on inactivated or live attenuated viruses.
Inactivated vaccines (whole virus, split virion, or surface antigen) are
administered by intramuscular or intradermal injection, whereas live
vaccines are administered intranasally. The invention can be used with
all of these vaccine forms.

[0118] Some embodiments of the invention use a surface antigen influenza
vaccine (inactivated). Such vaccines contain fewer viral components than
a split or whole virion vaccine. They include the surface antigens
hemagglutinin and, typically, also neuraminidase. Processes for preparing
these proteins in purified form from influenza viruses are well known in
the art. The FLUVIRIN®, AGRIPPAL® and INFLUVAC® products are
examples of surface antigen influenza vaccines.

[0119] Where the invention uses a surface antigen influenza vaccine, this
virus may have been grown in eggs or in cell culture (see below). The
current standard method for influenza virus growth for vaccines uses
embryonated SPF hen eggs, with virus being purified from the egg contents
(allantoic fluid). If egg-based viral growth is used then one or more
amino acids may be introduced into the allantoid fluid of the egg
together with the virus [24]. Virus is first grown in eggs. It is then
harvested from the infected eggs. Virions can be harvested from the
allantoic fluid by various methods. For example, a purification process
may involve zonal centrifugation using a linear sucrose gradient solution
that includes detergent to disrupt the virions. Antigens may then be
purified, after optional dilution, by diafiltration. Chemical means for
inactivating a virus include treatment with an effective amount of one or
more of the following agents: detergents, formaldehyde,
β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60),
binary ethylamine, acetyl ethyleneimine, or combinations thereof.
Non-chemical methods of viral inactivation are known in the art, such as
for example UV light or gamma irradiation.

[0120] Some embodiments of the invention can use whole virus, split virus,
virosomes, live attenuated virus, or recombinant hemagglutinin. These
vaccines can easily be distinguished from surface antigen vaccines by
testing their antigens e.g. for the presence of extra influenza virus
proteins.

[0121] Whole inactivated virus can be obtained by harvesting virions from
virus-containing fluids (e.g. obtained from eggs or from culture medium)
and then treating them as described above.

[0122] Split virions are obtained by treating purified virions with
detergents (e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl
phosphate, Triton X-100, Triton N101, cetyltrimethylammonium bromide,
Tergitol NP9, etc.) to produce subvirion preparations, including the
`Tween-ether` splitting process. Methods of splitting influenza viruses,
for example are well known in the art e.g. see refs. 19-24, etc.
Splitting of the virus is typically carried out by disrupting or
fragmenting whole virus, whether infectious or non-infectious with a
disrupting concentration of a splitting agent. The disruption results in
a full or partial solubilisation of the virus proteins, altering the
integrity of the virus. Preferred splitting agents are non-ionic and
ionic (e.g. cationic) surfactants e.g. alkylglycosides,
alkylthioglycosides, acyl sugars, sulphobetaines, betains,
polyoxyethylene-alkylethers, N,N-dialkyl-Glucamides, Hecameg,
alkylphenoxy-polyethoxyethanols, NP9, quaternary ammonium compounds,
sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butyl
phosphate, myristyltrimethylammonium salts, lipofectin, lipofectamine,
and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols (e.g. the Triton
surfactants, such as Triton X-100 or Triton N101), polyoxyethylene
sorbitan esters (the Tween surfactants), polyoxyethylene ethers,
polyoxyethlene esters, etc. One useful splitting procedure uses the
consecutive effects of sodium deoxycholate and formaldehyde, and
splitting can take place during initial virion purification (e.g. in a
sucrose density gradient solution). Thus a splitting process can involve
clarification of the virion-containing material (to remove non-virion
material), concentration of the harvested virions (e.g. using an
adsorption method, such as CaHPO4 adsorption), separation of whole
virions from non-virion material, splitting of virions using a splitting
agent in a density gradient centrifugation step (e.g. using a sucrose
gradient that contains a splitting agent such as sodium deoxycholate),
and then filtration (e.g. ultrafiltration) to remove undesired materials.
Split virions can usefully be resuspended in sodium phosphate-buffered
isotonic sodium chloride solution. Examples of split, vaccines are the
BEGRIVAC®, INTANZA®, FLUARIX®, FLUZONE® and FLUSHIELD®
products.

[0123] Virosomes are nucleic acid free viral-like liposomal particles
[25]. They can be prepared by solubilization of virus with a detergent
followed by removal of the nucleocapsid and reconstitution of the
membrane containing the viral glycoproteins. An alternative method for
preparing virosomes involves adding viral membrane glycoproteins to
excess amounts of phospholipids, to give liposomes with viral proteins in
their membrane.

[0124] Live attenuated viruses are obtained from viruses (grown in eggs or
in cell culture), but the viruses are not inactivated. Rather, the virus
is attenuated ("att") e.g so as not to produce influenza-like illness in
a ferret model of human influenza infection. It may also be a
cold-adapted ("ca") strain i.e. it can replicate efficiently at
25° C., a temperature that is restrictive for replication of many
wildtype influenza viruses. It may also be temperature-sensitive ("ts")
i.e. its replication is restricted at temperatures at which many
wild-type influenza viruses grow efficiently (37-39° C.). The
cumulative effect of the ca, ts, and att phenotype is that the virus in
the attenuated vaccine can replicate in the nasopharynx to induce
protective immunity in a typical human patient, but it does not cause
disease i.e. it is safe for general administration to the target human
population. These viruses can be prepared by purifying virions from
virion-containing fluids e.g. after clarification of the fluids by
centrifugation, then stabilization with buffer (e.g. containing sucrose,
potassium phosphate, and monosodium glutamate). Live vaccines include the
FLUMIST® product. Although live vaccines can be used with the
invention, it is preferred to use non-live vaccines.

[0125] As an alternative to using antigens obtained from virions,
haemagglutinin can be expressed in a recombinant host (e.g. in an insect
cell line, such as Sf9, using a baculovirus vector) and used in purified
form [26-28] or in the form of virus-like particles (VLPs; e.g. see
references 29 & 30).

[0126] Some embodiments of the invention use influenza vaccine prepared
from viruses which were grown in cell culture, rather than in eggs. When
cell culture is used, the viral growth substrate will typically be a cell
line of mammalian origin. Suitable mammalian cells of origin include, but
are not limited to, hamster, cattle, primate (including humans and
monkeys) and dog cells. Various cell types may be used, such as kidney
cells, fibroblasts, retinal cells, lung cells, etc. Examples of suitable
hamster cells are the cell lines having the names BHK21 or HKCC. Suitable
monkey cells are e.g. African green monkey cells, such as kidney cells as
in the Vero cell line. Suitable dog cells are e.g. kidney cells, as in
the MDCK cell line. Thus suitable cell lines include, but are not limited
to: MDCK; CHO; 2931; BHK; Vero; MRC-5; PER.C6; WI-38; etc. Preferred
mammalian cell lines for growing influenza viruses include: MOCK cells
[31-34], derived from Madin Darby canine kidney; Vero cells [35-37],
derived from African green monkey (Cercopithecus aethiops) kidney; or
PER.C6 cells [38], derived from human embryonic retinoblasts. These cell
lines are widely available e,g from the American Type Cell Culture (ATCC)
collection, from the Coriell Cell Repositories, or from the European
Collection of Cell Cultures (ECACC). For example, the ATCC supplies
various different Vero cells under catalog numbers CCL-81, CCL-81.2,
CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog number
CCL-34. PER.C6 is available from the ECACC under deposit number 96022940.
As a less-preferred alternative to mammalian cell lines, virus can be
grown on avian cell lines [e.g. refs. 39-41], including cell lines
derived from ducks (e.g. duck retina) or hens. Examples of avian cell
lines include avian embryonic stem cells [39,42] and duck retina cells
[40]. Suitable avian embryonic stem cells, include the EBx cell line
derived from chicken embryonic stem cells, E1345, EB14, and EB14-074
[43]. Chicken embryo fibroblasts (CEF) may also be used.

[0127] The most preferred cell lines for growing influenza viruses are
MDCK cell lines. The original MDCK cell line is available from the ATCC
as CCL-34, but derivatives of this cell line may also be used. For
instance, reference 31 discloses a MDCK cell line that was adapted for
growth in suspension culture (`MDCK 33016`, deposited as DSM ACC 2219).
Similarly, reference 44 discloses a MDCK-derived cell line that grows in
suspension in serum-free culture (`B-702`, deposited as FERM BP-7449).
Reference 45 discloses non-tumorigenic MDCK cells, including `MDCK-S`
(ATCC PTA-6500), `MDCK-SF101` (ATCC PTA-6501), `MDCK-SF102` (ATCC
PTA-6502) and `MDCK-SF103` (PTA-6503), Reference 46 discloses MDCK cell
lines with high susceptibility to infection, including `MDCK.5F1` cells
(ATCC CRL-12042). Any of these MDCK cell lines can be used.

[0128] Where virus has been grown on a mammalian cell line then products
of the invention will advantageously be free from egg proteins (e.g.
ovalbumin and ovomucoid) and from chicken DNA, thereby reducing potential
allergenicity.

[0129] Hemagglutinin in cell-derived products of the invention can have a
different glycosylation pattern from the patterns seen in egg-derived
viruses. Thus the HA (and other glycoproteins) may include glycoforms
that are not seen in chicken eggs. Useful HA includes canine glycoforms.

[0130] The absence of egg-derived materials and of chicken glycoforms
provides a way in which vaccine prepared from viruses grown in cell
culture can be distinguished from egg-derived products.

[0131] Where virus has been grown on a cell line then the culture for
growth, and also the viral inoculum used to start the culture, will
preferably be free from (i.e. will have been tested for and given a
negative result for contamination by) herpes simplex virus, respiratory
syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus,
rhinovirus, reoviruses, polyomaviruses, birnaviruses, circoviruses,
and/or parvoviruses [47]. Absence of herpes simplex viruses is
particularly preferred.

[0132] For growth on a cell line, such as on MDCK cells, virus may be
grown on cells in suspension [31, 48, 49] or in adherent culture. One
suitable MDCK cell line for suspension culture is MDCK 33016 (deposited
as DSM ACC 2219). As an alternative, microcarrier culture can be used.

[0133] Cell lines supporting influenza virus replication are preferably
grown in serum-free culture media and/or protein free media. A medium is
referred to as a serum-free medium in the context of the present
invention in which there are no additives from serum of human or animal
origin. Protein-free is understood to mean cultures in which
multiplication of the cells occurs with exclusion of proteins, growth
factors, other protein additives and non-serum proteins, but can
optionally include proteins such as trypsin or other proteases that may
be necessary for viral growth. The cells growing in such cultures
naturally contain proteins themselves.

[0135] The method for propagating virus in cultured cells generally
includes the steps of inoculating the cultured cells with the strain to
be cultured, cultivating the infected cells for a desired time period for
virus propagation, such as for example as determined by virus titer or
antigen expression (e.g. between 24 and 168 hours after inoculation) and
collecting the propagated virus. The cultured cells are inoculated with a
virus (measured by PFU or TCID50) to cell ratio of 1:500 to 1:1,
preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The virus is added
to a suspension of the cells or is applied to a monolayer of the cells,
and the virus is absorbed on the cells for at least 60 minutes but
usually less than 300 minutes, preferably between 90 and 240 minutes at
25° C. to 40° C., preferably 28° C. to 37° C.
The infected cell culture (e.g. monolayers) may be removed either by
freeze-thawing or by enzymatic action to increase the viral content of
the harvested culture supernatants. The harvested fluids are then either
inactivated or stored frozen. Cultured cells may be infected at a
multiplicity of infection ("m.o.i.") of about 0.0001 to 10, preferably
0.002 to 5, more preferably to 0.001 to 2. Still more preferably, the
cells are infected at a m.o.i of about 0.01. Infected cells may be
harvested 30 to 60 hours post infection. Preferably, the cells are
harvested 34 to 48 hours post infection. Still more preferably, the cells
are harvested 38 to 40 hours post infection. Proteases (typically
trypsin) are generally added during cell culture to allow viral release,
and the proteases can be added at any suitable stage during the culture.

[0136] A vaccine product including vaccine prepared from cell culture
preferably contains less than 10 ng (preferably less than 1 ng, and more
preferably less than 100 pg) of residual host cell DNA per dose, although
trace amounts of host cell DNA may be present.

[0137] It is preferred that the average length of any residual host cell
DNA is less than 500 bp e.g. less than 400 bp, less than 300 bp, less
than 200 bp, less than 100 bp, etc.

[0138] Contaminating DNA can be removed during vaccine preparation using
standard purification procedures e.g. chromatography, etc. Removal of
residual host cell DNA can be enhanced by nuclease treatment e.g. by
using a DNase. A convenient method for reducing host cell DNA
contamination is disclosed in references 51 & 52, involving a two-step
treatment, first using a DNase (e.g. Benzonase), which may be used during
viral growth, and then a cationic detergent (e.g. CTAB), which may be
used during virion disruption. Treatment with an alkylating agent, such
as β-propiolactone, can also be used to remove host cell DNA, and
advantageously may also be used to inactivate virions [53].

[0139] Some embodiments of the invention use a monovalent influenza
vaccine (i.e. it includes hemagglutinin antigen from a single influenza
virus strain) but in some embodiments it may be a multivalent vaccine,
such as a bivalent vaccine, trivalent vaccine, a tetravalent vaccine, or
a >4-valent vaccine (i.e. including hemagglutinin from more than four
different influenza virus strains). Monovalent and multivalent vaccines
are readily distinguished by testing for multiple HA types, by amino acid
sequencing, etc.

[0140] A monovalent vaccine is particularly useful for immunising against
a pandemic or potentially-pandemic strain, either during a pandemic or in
a pre-pandemic situation. Characteristics of these strains are: (a) they
contain a new hemagglutinin compared to the hemagglutinins in
currently-circulating human strains, i.e. one that has not been evident
in the human population for over a decade (e.g. H2), or has not
previously been seen at all in the human population (e.g. H5, H6 or H9,
that have generally been found only in bird populations), such that the
human population will be immunologically naive to the strain's
hemagglutinin; (b) they are capable of being transmitted horizontally in
the human population; and (c) they are pathogenic to humans. These
strains may have any of influenza A HA subtypes H1, H2, H3, H4, H5, H6,
H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. A virus with H5
hemagglutinin type is preferred for immunizing against pandemic
influenza, or a H2, H7 or H9 subtype. The invention may protect against
one or more of influenza A virus NA subtypes N1, N2, N3, N4, N5, N6, N7,
N8 or N9. Thus possible strains include H5N1, H5N3, H9N2, H2N2, H7N1 and
H7N7, and any other emerging potentially pandemic strains.

[0141] A multivalent vaccine is more typical in a seasonal setting e.g. a
trivalent vaccine is typical, including hemagglutinins from two influenza
A virus strains and one influenza B virus strain, such as from a H1N1
influenza A strain, a H3N2 influenza A virus strain, and an influenza B
virus strain. A tetravalent vaccine is also useful [54] e.g. including
antigens from two influenza A virus strains and two influenza 13 virus
strains, or three influenza A virus strains and one influenza B virus
strain. Thus a vaccine may be bivalent, trivalent, tetravalent, etc.
Except for monovalent vaccines, it is usual to include hemagglutinin from
both influenza A and influenza B virus strains. In vaccines including
only two influenza A virus strains, these will usually be one H1 strain
(e.g. a H1N1 strain) and one H3 strain (e.g. a H3N2 strain). In some
embodiments, however, there may be one pandemic influenza A virus strain
and one H1 strain, or one pandemic influenza A virus strain and one H3
strain.

[0142] Where a vaccine includes more than one strain of influenza, the
different strains are typically grown separately and are mixed after the
viruses have been harvested and antigens have been prepared. Thus a
process of the invention may include the step of mixing antigens from
more than one influenza strain.

[0143] As described in reference 54, exemplary tetravalent vaccines can
include hemagglutinin from two influenza A virus strains and two
influenza B virus strains (`A-A-B-B`), or from three influenza A virus
strains and one influenza B virus strain (`A-A-A-B`).

[0144] Influenza B virus currently does not display different HA subtypes,
but influenza B virus strains do fall into two distinct lineages. These
lineages emerged in the late 1980s and have HAs which can be
antigenically and/or genetically distinguished from each other [55].
Current influenza B virus strains are either B/Victoria/2/87-like or
B/Yamagata/16/88-like. Where a vaccine of the invention includes two
influenza B strains, this will usually be one B/Victoria/2/87-like strain
and one B/Yamagata/16/88-like strain. These strains are usually
distinguished antigenically, but differences in amino acid sequences have
also been described for distinguishing the two lineages e.g.
B/Yamagata/16/88-like strains often (but not always) have HA proteins
with deletions at amino acid residue 164, numbered relative to the
`Lee40` HA sequence [56].

[0146] In vaccines including three influenza A virus strains, these will
usually be one HI strain (e.g. a H1N1 strain) and two H3 strains (e.g.
two H3N2 strains). The two H3 strains will have antigenically distinct HA
proteins e.g. one H3N2 strain that cross-reacts with A/Moscow/10/99 and
one H3N2 strain that cross-reacts with A/Fujian/411/2002. The two H3
strains may be from different clades (clades A, B and C of H3N2 strains
are disclosed in reference 57). In some embodiments, however, one of
these strains (i.e. H1, or one of the two H3 strains) may be replaced by
a pandemic strain.

[0147] Thus one preferred A-A-A-B vaccine includes hemagglutinins from:
(i) a H1N1 strain; (ii) a A/Moscow/10/99-like H3N2 strain; (iii) a
A/Fujian/411/2002-like H3N2 strain; and (iv) an influenza B virus strain,
which may be B/Victoria/2/87-like or B/Yamagata/16/88-like.

[0149] Another preferred A-A-A-B vaccine includes hemagglutinins from: (i)
two different H1 strains, (ii) a H3N2 strain, and (iii) an influenza B
strain.

[0150] Where antigens are present from two or more influenza B virus
strains, at least two of the influenza B virus strains may have distinct
hemagglutinins but related neuraminidases. For instance, they may both
have a B/Victoria/2/87-like neuraminidase [58] or may both have a
B/Yamagata/16/88-like neuraminidase. For instance, two
B/Victoria/2/87-like neuraminidases may both have one or more of the
following sequence characteristics: (I) not a serine at residue 27, but
preferably a leucine; (2) not a glutamate at residue 44, but preferably a
lysine; (3) not a threonine at residue 46, but preferably an isoleucine;
(4) not a proline at residue 51, but preferably a serine; (5) not an
arginine at residue 65, but preferably a histidine; (6) not a glycine at
residue 70, but preferably a glutamate; (7) not a leucine at residue 73,
but preferably a phenylalanine; and/or (8) not a proline at residue 88,
but preferably a glutamine. Similarly, in some embodiments the
neuraminidase may have a deletion at residue 43, or it may have a
threonine; a deletion at residue 43, arising from a trinucleotide
deletion in the NA gene, has been reported as a characteristic of
B/Victoria/2/87-like strains, although recent strains have regained
Thr-43 [58]. Conversely, of course, the opposite characteristics may be
shared by two B/Yamagata/16/88-like neuraminidases e.g. S27, E44, 146,
P51, R65, G70, L73, and/or P88. These amino acids are numbered relative
to the `Lee40` neuraminidase sequence [59]. Thus a A-A-B-B vaccine of the
invention may use two B strains that are antigenically distinct for HA
(one B/Yamagata/16/88-like, one B/Victoria/2/87-like), but are related
for NA (both B/Yamagata/16/88-like, or both B/Victoria/2/87-like).

[0151] In some embodiments, the invention does not encompass a trivalent
split vaccine containing hemagglutinin from each of A/New Caledonia/20/99
(H1N1), A/Wyoming/03/2003 (H3N2) and B/Jiangsu/10/2003 strains.

[0152] Strains whose antigens can usefully be included in the compositions
include strains which are resistant to antiviral therapy (e.g. resistant
to oseltamivir [60] and/or zanamivir), including resistant pandemic
strains [61].

[0153] In some embodiments of the invention, a vaccine may include a small
amount of mercury-based preservative, such as thiomersal or merthiolate.
When present, such preservatives will typically provide less than 5
μg/ml mercury, and lower levels are possible e.g. <1 μg/ml,
<0.5 μg/ml. Preferred vaccines are free from thiomersal, and are
more preferably mercury-free [23,62]. Such vaccines may include a
non-mercurial preservative. Non-mercurial alternatives to thiomersal
include 2-phenoxyethanol or α-tocopherol succinate [23]. Most
preferably, a vaccine is preservative-free.

[0154] In some embodiments, a vaccine may include a stabilising amount of
gelatin e.g. at less than 0.1%. In other embodiments, however, a vaccine
is gelatin-free. The absence of gelatin can assure that the vaccine is
safe in the small proportion of patients who are gelatin-sensitive
[63,64].

[0155] In some embodiments, a vaccine may include one or more antibiotics
e.g. neomycin, kanamycin, polymyxin B. In preferred embodiments, though,
the vaccine is free from antibiotics.

[0156] In some embodiments, a vaccine may include formaldehyde. In
preferred embodiments, though, the vaccine is free from formaldehyde.

[0157] As mentioned above, in some embodiments a vaccine may include egg
components (e.g. ovalburnin and ovomucoid), but preferred embodiments are
free from egg components.

[0158] The preparation of vaccines without the use of certain components
and additives is disclosed in reference 65, thereby ensuring that these
materials are not present even in residual amounts.

[0159] Hemagglutinin (HA) is the main immunogen in current inactivated
influenza vaccines, and vaccine doses are standardised by reference to HA
levels, typically measured by SRID. Existing vaccines typically contain
about 15 μg of HA per strain, although lower doses can be used e.g.
for children, or in pandemic situations, or when using an adjuvant.
Fractional doses such as 1/2 (i.e. 7.5 μg HA per strain), 1/4 and 1/8
have been used, as have higher doses (e.g. 3× or 9× doses
[66,67]). These vaccines have a dosage volume of 0.5 ml i.e. a typical HA
concentration of 30 μg/ml/strain. The trivalent INTANZA® product
contains 9 μg of HA per strain in a 0.1 ml volume i.e. a HA
concentration of 90 μg/ml/strain, giving a total HA concentration of
270 μg/ml.

[0160] Products of the present invention can include between 0.1 and 50
μg of HA per influenza strain per dose, preferably between 0.1 and 50
μg e.g. 1-20 μg. Ideally a product has ≦16 μg
hemagglutinin per strain e,g 1-15 μg, 1-10 μg, 1-7.5 μg, 1-5
μg, etc. Particular HA doses per strain include e.g. about 15, about
10, about 7.5, about 5, about 3.8, about 1.9, about 1.5, etc.

[0161] For live vaccines, dosing is measured by median tissue culture
infectious dose (TCID50) rather than HA content e.g. a TCID50
of between 106 and 108 (preferably between
106.5-107.5) per strain per dose.

[0162] Influenza strains used with the invention may have a natural HA as
found in a wild-type virus, or a modified HA. For instance, it is known
to modify HA to remove determinants (e.g. hyper-basic regions around the
HA1/HA2 cleavage site) that cause a virus to be highly pathogenic in
avian species. The use of reverse genetics facilitates such
modifications.

[0163] Vaccine products of the invention can include components in
addition to the influenza vaccine antigens. As discussed above, for
example, they can include a biosoluble and biodegradable matrix material,
or an oral film polymer.

[0164] Vaccine products may include a detergent. The level of detergent
can vary widely e.g. between 0.05-50 μg detergent per pg of HA
(`μg/μg`). A low level of detergent can be used e.g. between 0.1-1
μg/μg, or a high level can be used e.g. between 5-30 μg/μg.
The detergent may be a single detergent (e.g. polysorbate 80, or CTAB) or
a mixture (e.g. both polysorbate 80 and CTAB). Preferred detergents are
non-ionic, such as polysorbate 80 (`Tween 80`) or octyl phenol ethoxylate
(`Triton X100`). Polysorbate 80 may be present at between 0.05-50 μg
polysorbate 80 per pg of HA e.g. between 0.1-1 μg/μg, 0.1-0.8
μg/μg, 0.1-0.5 μ/μg, 5-40 μg/μg, 5-30 μg/μg, or
8-25 μg/μg.

[0165] As mentioned above, some vaccine products may include preservatives
such as thiomersal or 2-phenoxyethanol, but preferred vaccines are
mercury- or preservative-free.

[0166] Vaccine products may include a physiological salt, such as a sodium
salt. Sodium chloride (NaCl) is preferred, which may be present at
between 1 and 20 mg/ml. Other salts that may be present include potassium
chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate,
magnesium chloride, calcium chloride, etc.

[0167] Vaccine products may include one or more buffers. Typical buffers
include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate
buffer; a histidine buffer (particularly with an aluminum hydroxide
adjuvant); or a citrate buffer. Buffers will typically be included in the
5-20 mM range.

[0171] Influenza vaccines are currently recommended for use in pediatric
and adult immunisation, from the age of 6 months. Thus a human subject
may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years
old, or at least 55 years old. Preferred subjects for receiving the
vaccines are the elderly (e.g. ≧50 years old, ≧60 years
old, and preferably ≧65 years), the young (e.g. ≦5 years
old), hospitalised subjects, healthcare workers, armed service and
military personnel, pregnant women, the chronically immunodeficient
subjects, subjects who have taken an antiviral compound (e.g. an
oseltamivir or zanamivir compound; see below) in the 7 days prior to
receiving the vaccine, people with egg allergies and people travelling
abroad. The vaccines are not suitable solely for these groups, however,
and may be used more generally in a population. For pandemic strains,
administration to all age groups is preferred.

[0172] Administration of more than one dose (typically two doses) is
particularly useful in immunologically naive patients e.g. for people who
have never received an influenza vaccine before, or for vaccinating
against a new HA subtype (as in a pandemic outbreak).

[0173] Reconstitution Using a Buffer

[0174] In a further aspect, the invention provides a process for preparing
a vaccine antigen, comprising steps of (i) increasing the concentration
of an antigen in a liquid composition including that antigen, to provide
a concentrated antigen, (ii) lyophilising the concentrated antigen, to
provide the lyophilised vaccine antigen, and (iii) reconstituting the
lyophilised vaccine antigen in an aqueous buffer to provide a
reconstituted antigen.

[0175] Apart from the techniques which can be used for concentration in
step (i), and the material which is used for reconstitution in step
(iii), details for this further aspect are the same as already described
herein.

[0176] In contrast to the preceding aspects of the invention, step (i) is
not restricted to using centrifugal filtration and/or ultrafiltration in
step (i). Various techniques can be used for concentration step (i),
including but not limited to: centrifugal filtration; ultrafiltration; or
tangential flow filtration (also known as crossflow filtration). These
three concentration techniques are not mutually exclusive e.g. the
invention can use tangential flow ultrafiltration.

[0177] Tangential flow filtration (TFF) involves passing a liquid
tangentially across a filter membrane. The sample side is typically held
at a positive pressure relative to the filtrate side. As the liquid flows
over the filter, components therein can pass through the membrane into
the filtrate. Continued flow causes the volume of the filtrate to
increase, and thus the concentration of the antigen in the retentate
increases. TFF contrasts with deadend filtration, in which sample is
passed through a membrane rather than tangentially to it. Many TFF
systems are commercially available. The MWCO of a TFF membrane determines
which solutes can pass through the membrane (i.e. into the filtrate) and
which are retained (i.e. in the retentate). The MWCO of a TFF filter used
with the invention will be selected such that substantially all of the
antigen of interest remains in the retentate.

[0179] In this aspect, reconstitution in step (iii) uses a buffer (e.g. a
phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a
histidine buffer, or a citrate buffer). Buffers will typically be
included in the 5-20 mM range. A phosphate buffer is preferred.
Reconstitution using water, or using a non-aqueous solvent, is not part
of this aspect of the invention.

[0180] Step (i) concentrated the a first liquid volume of vaccine antigen,
providing a composition with the same amount of antigen in a second
(reduced) liquid volume. Step (ii) dried this concentrated material. This
dried material is reconstituted in a third volume of buffer. The second
volume is lower than the first volume. The third volume is either equal
to or, preferably, less than the second volume (and thus, by definition,
lower than the first volume i.e. the overall process has provided a more
concentrated aqueous form of the antigen in the initial liquid
composition).

[0181] The reconstituted antigen of this aspect can be used to formulate
vaccine as already described herein.

[0183] In one embodiment the buffer-reconstituted antigen is used to coat
microneedles as described above.

[0184] General

[0185] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist exclusively of
X or may include something additional e.g. X+Y.

[0186] The word "substantially" does not exclude "completely" e.g. a
composition which is "substantially free" from Y may be completely free
from Y. Where necessary, the word "substantially" may be omitted from the
definition of the invention.

[0187] The term "about" in relation to a numerical value x is optional and
means, for example, x±5%.

[0188] Unless specifically stated, a process comprising a step of mixing
two or more components does not require any specific order of mixing.
Thus components can be mixed in any order. Where there are three
components then two components can be combined with each other, and then
the combination may be combined with the third component, etc.

[0189] Where animal (and particularly bovine) materials are used in the
culture of cells, they should be obtained from sources that are free from
transmissible spongiform encephalopathies (TSEs), and in particular free
from bovine spongiform encephalopathy (BSE). Overall, it is preferred to
culture cells in the total absence of animal-derived materials.

[0190] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.

[0193] Centrifugal filtration used a Millipore® device with a 10 kDa
cut-off, operated at 5000 rpm.

[0194] Three centrifugation durations were tested: 15, 30 and 45 minutes.
The retentate (concentrate) and filtrate were checked to see the location
of an influenza virus hemagglutinin. FIG. 1 shows that the antigen is
still in the retentate after 45 minutes. Antigen concentration was 3-fold
after 15 minutes, 6-fold after 30 minutes, and 13-fold after 45 minutes.
Antigen recovery was 40% after 15 minutes, 41% after 30 minutes, and 55%
after 45 minutes. Thus 45 minutes was chosen for further work.

[0195] In further work, antigen was lyophilised after centrifugation, to
provide further concentration. Sucrose was used as the lyoprotectant,
alone (at two different concentrations) or with mannitol. Lyophilised
material was reconstituted. The reconstituted samples contained visible
aggregates. Relative to the starting material, HA content (measured by
ELISA) was concentrated as follows:

[0196] Thus the combination of centrifugation and lyophilisation can
provide a >25-fold concentration in influenza virus HA content. The
two centrifuged samples were also assessed by SRID and they showed a
21.1× and 35.1× increase in HA content, with the higher
sucrose level again giving better results.

[0197] Ultrafiltration

[0198] Ultrafiltration used an Amicon® stir cell concentrator with a 10
kDa cut-off membrane made from regenerated cellulose, operated under
pressurised nitrogen for 1 hour.

[0199] If a lyophilisation was added, followed by reconstitution back into
the pre-lyophilisation volume, the reconstituted material had a HA
concentration (as measured by SRID) comparable to the starting material,
indicating no loss of functional antigen. The reconstituted material was
stable for >2 weeks.

[0200] It will be understood that the invention has been described by way
of example only and modifications may be made whilst remaining within the
scope and spirit of the invention.